Antimitotic compounds interfere with the dynamiic assembly and disassembly, of xcex1-and xcex2-tubulin into microtubules causing cells to arrest in mitosis. Prolonged arrest in mitosis eventually leads to cell death, often by apoptosis. Two chemical classes of antimitotic agents, the vinca alkaloids (vinblastine, vincristine, and vinorelbine) and the taxanes (paclitaxel and docetaxel), are clinically useful anticancer drugs. Most known antinmitotic agents induce mitotic arrest by inhibiting the polymerization of tubulin into microtubules. This is the mechanism of the vinca alkaloids and rhizoxin.
Paclitaxel was the first chemical entity shown to cause mitotic arrest by stabilzing microtubules against depolymerization. Four additional chemotypes that have paclitaxel-like effects were later identified. These include the myxobacterium metabolites epothilones A and B, the marine sponge metabolites discodermolide, laulimalide, and isolaulimalide, and the soft coral terpenoid, eleutherobin (shown below as Compound 1.) Ojirna et al. (1999) Proc. Natl. Acad. Sci. USA 96:4256-4261, propose a common pharmacophore for the microtubule stabilizing compounds that effectively accommodates nonataxel, paclitaxel, discodermolide, eleutherobin, and the epothilones. This model predicts that three regions of eleutherobin (boxes A, B, and C below) are important for binding to tubulin (Me=methyl; Ac=acetyl). 
The majority of known antimitotic natural products were initially isolated because they exhibited potent in vitro cytotoxicity. Only subsequent detailed mechanism of action studies revealed that they arrested cells in mitosis and interfered with tubulin assembly and disassembly dynamics. For example, rhizoxin is a 16-membered ring macrolide first isolated in 1984 and determined to be very cytotoxic. Only later was rhizoxin shown to cause the accumulation of cells in mitosis. Sarcodictyins A-D were the first members of a cytotoxic terpenoid class of compounds to be identified (see: D""Ambrosio, M., et al. (1987) Helv. Chim. Acta. 70:2019-2027; and, (1988) Helv. Chim. Acta. 71:964-976), their paclitaxel-like properties being recognized only later.
Eleutherobin, a diterpene glycoside, was originally isolated from the soft coral Eleutherobia sp. (possibly E. albiflora) collected in Western Australia (see: Lindel, T. et al. (1997) J. Am. Chem. Soc. 119:8744-8745; and, international patent application published May 23, 1996 under WO 96/14745). Subsequently, eleuthosides A and B were isolated from a different species of Eleutherobia (E. aurea). The eleuthosides differ from eleutherobin by the presence of a hydroxyl substitute at the C-4 position shown above (rather than a methoxyl substitute) and, as shown above, by the presence of an acetyl group at the 3xe2x80x3 or the 4xe2x80x3 position of the arabinose moiety shown above, in addition to an acetyl at the 2xe2x80x3 position (Ketzinel, S., et al. (1996) J. Nat. Prod. 59:873-875). Later, a total synthesis of eleutherobin and eleuthosides A and B was reported (Nicolaou, K. C., et al. (1998) J. Am. Chem. Soc. 120:8674-8680). As reported in the latter reference, the eleuthosides may be made by converting C-4 ketal precursors to C-4 hydroxyl forms.
Using a new cell-based antimitotic assay, the inventors herein have demonstrated potent antimitotic activity in extracts of marine organisms providing abundant new sources of antimitotic terpenoids. Microscopic examination of cells arrested in mitosis by the extracts show tubulin bundling, similar to the effects of paclitaxel. Bioassay guided fractionation of extracts of marine organisms has led to the isolation of eleutherobin 1 and the novel diterpenes shown below, including desmethyleleutherobin 2, desacetyleleutherobin 3, isoeleutherobin A 4, Z-cleutherobin 5, carnbaeoside 6, and caribaeolin 7. 
This invention provides the use of organisms of the order Gorgonacea as a source for the preparation of purified or partially purified antimitotic compounds, including terpenoids. This invention provides a method to obtain antimitotic terpenoids wherein an extract of an organism of the order Gorgonacea in a solvent is subjected to fractionation to separate antimitotic compounds from compounds lacking antimitotic activity. Fractionation may include any suitable process for separation of terpenoid compounds. Antimitotic terpenoids may comprise one or more of the compounds identified as Compounds 1-7 above as well as sarcodictyin A. Preferably the organism employed is a gorgonian coral such as Erythropodium caribaeorum. 
A solvent used for preparing extracts of organisms according to this invention may be any suitable solvent for extraction or dissolution of terpenoids, including alcohols (e.g. methanol, ethanol), acetone, acetate compounds, chloroform, dichloromethane, etc. Mixtures of polar solvents with water may be used, the ratios to be determined by procedures known in the art. In some applications, it will be preferred that the solvent be one that is incapable of forming a ketal compound, which excludes the alcohols. A particularly preferred solvent is an acetate such as ethyl acetate (EtOAc).
Preferred fractionation procedures are chromatographic. Preferably, several chromatography procedures will be performed, with each procedure intended to separate compounds according to differing parameters such as: solubility (e.g. gradient elution), and molecular size (e.g. by use of a molecular sieve such as a Sephadex(trademark) gel). A suitable gradient elution chromatography procedure involves elution of compounds from a substrate (e.g. a silica bed in a column) by application of mixed solvents having varying ratios of solvent components (e.g. reversed or normal phase; vacuum or flash liquid chromatography). For example, applied solvents may have varying ratios of a polar solvent (e.g. methanol: MeOH) to either: a different polar solvent (e.g. EtOAc or H2O), or a non-polar solvent (e.g. hexane). Selection of appropriate bed substrates and elution profiles as well as chromatography bed design may be done using standard laboratory procedures and protocols, or the specific procedures described herein may be employed. Purification may also be accomplished by using high pressure liquid chromatography (HPLC) which may be used to particular advantage as a final step in purification. In some cases, purification by crystallization of compounds from solution may be accomplished.
Fractionation of compounds in this invention may be guided by monitoring for particular chemical or physical characteristics of desired or undesired compounds. Monitoring for the specific characteristics of such compounds as described herein may be carried out using standard procedures, such as determination of melting/decomposition temperature or by spectroscopic methods (including mass spectrometry, UV spectrometry and nuclear magnetic resonance (NMR)). For example, the unique UV chromophore of eleutherobin may be used to monitor the presence of that compound in fractions obtained as the method of this invention is carried out.
The method of this invention may also be guided by the use of any suitable test or assay for anrimitotic activity. Presence or absence of antimitotic compounds in crude extracts of selected organisms of the above-mentioned orders may be determined prior to the performance of the method of this invention. Further, such an assay may be used to monitor the presence of desired compounds in fractions obtained during performance of the method of this invention. Microscopic examination of cells treated with a test substance is a traditional test for antimitotic activity. Other suitable assays are disclosed herein.
This invention also provides an assay for antimitotic activity comprising:
(a) applying a sample to be tested for antimitotic activity to cells which are capable of mitosis in culture;
(b) culturing the cells for a time sufficient for the cells to undergo mitosis;
(c) fixing the cells on a substrate and treating the cells to increase the cells"" permeability to an antibody; and
(d) applying a mitotic cell-specific antibody to the cells and detecting binding of said antibody within the cells.
In the above-described assay, cells may be fixed using any suitable method for the type of cell and the substrate. Formaldehyde is a common fixative. Permeability may be increased by treatment in known ways, including treatment with an alcohol and/or a detergent. A preferred method of detecting binding of the antibody is to apply a second antibody capable of binding to the mitotic cell-specific antibody used in (d). The second antibody is typically linked to a detectable indicator. After removal of unbound antibodies from the cells, the presence of bound mitotic cell-specific antibody is detected by determining the presence of the detectable indicator. When the detectable indicator is an enzyme, its presence is determined by determining the presence of a product of the reaction that is catalyzed by the enzyme.
This invention also provides an antimitotic compound and pharmaceutical preparations thereof, wherein the compound has the formula: 
wherein Me is methyl; R1, R2 and R3 are independently selected from the group consisting of H and a C1-C6 acyl; R4 is selected from the group consisting of H2Me and a substituted or unsubstituted straight-chain, branched, or cyclic C1-C10 alkyl; providing that if R4 is Me, R1 is not acyl; and, providing that if R4 is H, two of R1, R2 and R3 are not acyl. Preferably, the acyl is acetyl and the alkyl is a C2-C5 straight-chain or branched moiety. A compound of this invention includes salts (preferably pharmaceutically acceptable salts) and also includes isomers, including those of the Z and E configurations and those of the xcex1 and xcex2 configurations at the glycosidic bond.
Preferred embodiments of this invention include those in which: R4 is H, ethyl, propyl, butyl or pentyl; one of R1 and R2 is H and the other Ac: and, R3 is H. In another preferred embodiment: R4 is Me; R1 and R3 are H; and, R2 is Ac.
A compound of this invention may be isolated from natural sources as described herein; prepared by total synthesis by adapting the methods of Nicolaou, K. C., et al. [supra]; or, from an intermediate. The intermediate may be prepared by total synthesis using conventional starting materials or obtained by reduction and glycosylation of sarcodictyin A (see: WO 96/14745). An intermediate used in the preparation of compounds of this invention may be isoeleutherobin A, desmethyleleutherobin or eleutherobin, with appropriate substitutions at R1-3 done using conventional procedures (such as the acetylation procedure described in the Examples below or by Nicolaou, K. C., et al. [supra]) and substitutions at C-4 done according to the methods described by Nicolaou, K. C., et al. [supra].
This invention also provides a method of converting a diterpenoid compound, including a compound of this invention having a hydroxyl substituent at C-4 to a compound having a ketal substituent at C-4 by contacting a compound having the hydroxyl substituent with an alcohol in the presence of a suitable acid catalyst. The catalyst may be an acid but is preferably a catalyst such as pyridinium p-toluenesulfonate. The alcohol may be methanol or any substituted or unsubstituted, straight-chain, branched, or cyclic alcohol having from 2-10 carbon atoms. Preferred alcohols are methanol, ethanol, the propanols, the butanols and the pentanols (to provide methoxyl, ethoxyl, propanoxyl, butanoxyl or pentanoxyl substituents respectively, at C-4.)
This invention also provides the use of a compound or a pharmaceutical preparation of this invention as an antimitotic agent and for the preparation of antimilotic medicaments. This invention also provides a method for causing mitotic arrest in one or more cells present in a cell population, comprising treating the cell population with a sufficient amount of a compound or pharmaceutically acceptable salt thereof, a pharmaceutical preparation or medicament of this invention to arrest mitosis in one or more cells in the cell population. The cell population may be a population of cancerous cells, including a tumor. This method may be performed in vitro or may be performed in vivo through administration to a human or animal patient with a cancer.